A single totipotent zygote has the remarkable ability to give rise to an entire multi-cellular organism. Methodologies to comprehensively map and modulate the parameters that govern this transformation will have far ranging impact on our understanding of development and also our ability to restore normal function in damaged or diseased tissues. While these processes have typically been studied in the context of model organisms, the advent of human pluripotent stem cells (hPSCs) has opened a powerful paradigm to recapitulate human development in ex vivo settings. Indeed hPSCs have been successfully differentiated to most somatic cell types of interest and recently also into complex three-dimensional organoids. These differentiation systems are beginning to offer an unprecedented insight into human biology, however two fundamental challenges have persisted: one, enabling efficacious differentiation of hPSCs to a mature phenotype that recapitulates an adult versus an embryonic or fetal tissue-of-origin like state; and two, generation of scalable and architecturally reproducible organotypic tissues. Focusing on liver differentiation from hPSCs, in this proposal we will explore the hypothesis that these two key objectives are intertwined, and achieving a mature phenotype is potentially linked to the establishment of an appropriate cellular niche. Towards this we will develop an integrated genome engineering and tissue engineering approach that will allow systematic manipulation of both genetic and cellular microenvironmental parameters during hPSC differentiation. Using this framework we hope to enable a deeper understanding of the interplay of gene regulatory networks and cellular niche on cell fate determination; and also potentially guide development of methodologies towards programming the processes of organogenesis for regenerative medicine applications.